Amidase

ABSTRACT

A purified thermostable enzyme is derived from the archael bacterium Thermococcus GU5L5. The enzyme has a molecular weight of about 68.5 kilodaltons and has cellulase activity. The enzyme can be produced from native or recombinant host cells and can be used for the removal of arginine, phenylalanine, or methionine amino acids from the N-terminal end of peptides in peptide or peptidomimetic synthesis. The enzyme is selective for the L, or ‘natural’ enantiomer of the amino acid derivatives and is therefore useful for the production of optically active compounds. These reactions can be performed in the presence of the chemically more reactive ester functionally, a step which is very difficult to achieve with nonenzymatic methods.

CROSS-RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/427,372, filed Oct. 25, 1999, which is a divisional of U.S.application Ser. No. 09/261,006, filed Mar. 2, 1999, now issued U.S.Pat. No. 6,004,796, which is Div. of Ser. No. 09/066,285, now U.S. Pat.No. 5,985,646, which is CON of Ser. No. 08/951,088, filed Oct. 15, 1997,now U.S. Pat. No. 6,136,583 which is a divisional of U.S. applicationSer. No. 08/664,646, filed Jun. 17, 1996, now issued U.S. Pat. No.5,877,001, the contents of which are hereby incorporated by reference.

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production and isolation of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention has been identified as an amidase and inparticular an enzyme having activity in the removal of arginine,phenylalanine or methionine from the N-terminal end of peptides inpeptide or peptidomimetic synthesis.

Thermophilic bacteria have received considerable attention as sources ofhighly active and thermostable enzymes (Bronneomeier, K. andStaudenbauer, W. L., D. R. Woods (Ed.), The Clostridia andBiotechnology, Butterworth Publishers, Stoneham, Mass. (1993). Recently,the most extremely thermophilic organotrophic eubacteria presently knownhave been isolated and characterized. These bacteria, which belong tothe genus Thermotoga, are fermentative microorganisms metabolizing avariety of carbohydrates (Huber, R. and Stetter, K. O., in Ballows, etal., (Ed.), The Procaryotes, 2nd Ed., Springer-Verlaz, New York, pgs.3809-3819 (1992)).

Because to date most organisms identified from the archaeal domain arethermophiles or hyperthermophiles, archaeal bacteria are also considereda fertile source of thermophilic enzymes.

In accordance with one aspect of the present invention, there isprovided a novel enzyme, as well as active fragments, analogs andderivatives thereof.

In accordance with another aspect of the present invention there areprovided isolated nucleic acid molecules encoding an enzyme of thepresent invention including mRNAs, DNAs, cDNAs, genomic DNAs as well asactive analogs and fragments of such enzymes.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a nucleic acid sequence encoding anenzyme of the present invention, under conditions promoting expressionof said enzyme and subsequent recovery of said enzyme.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such enzyme, or polynucleotideencoding such enzyme. The enzyme is useful for the removal of arginine,phenylalanine, or methionine amino acids from the N-terminal end ofpeptides in peptide or peptidomimetic synthesis. The enzyme is selectivefor the L, or “natural” enantiomer of the amino acid derivatives and istherefore useful for the production of optically active compounds. Thesereactions can be performed in the presence of the chemically morereactive ester functionality, a step which is very difficult to achievewith nonenzymatic methods. The enzyme is also able to tolerate hightemperatures (at least 70° C.), and high concentrations of organicsolvents (>40% DMSO), both of which cause a disruption of secondarystructure in peptides; this enables cleavage of otherwise resistantbonds.

In accordance with yet a further aspect of the present invention, thereis also provided nucleic acid probes comprising nucleic acid moleculesof sufficient length to specifically hybridize to a nucleic acidsequence of the present invention.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such enzymes, or polynucleotidesencoding such enzymes, for in vitro purposes related to scientificresearch, for example, to generate probes for identifying similarsequences which might encode similar enzymes from other organisms.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

FIGS. 1A, B, C, D and E are illustrations of the full-length DNA andcorresponding deduced amino acid sequence of the enzyme of the presentinvention. Sequencing was performed using a 378 automated DNA sequencer(Applied Biosystems, Inc.).

FIG. 2 shows the fluorescence versus concentration of DMSO. The filledand open boxes represent individual assays from Example 3.

FIG. 3 shows the relative initial linear rates (increase in fluorescenceper min. i.e. “activity”) versus concentration of DMF for the morereactive CBZ-L-arg-AMC, from Example 3.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

A coding sequence is “operably linked to” another coding sequence whenRNA polymerase will transcribe the two coding sequences into a singlemRNA, which is then translated into a single polypeptide having aminoacids derived from both coding sequences.

The coding sequences need not be contiguous to one another so long asthe expressed sequences are ultimately processed to produce the desiredprotein.

“Recombinant” enzymes refer to enzymes produced by recombinant DNAtechniques; i.e., produced from cells transformed by an exogenous DNAconstruct encoding the desired enzyme. “Synthetic” enzymes are thoseprepared by chemical synthesis.

A DNA “coding sequence of” or a “nucleotide sequence encoding” aparticular enzyme, is a DNA sequence which is transcribed and translatedinto an enzyme when placed under the control of appropriate regulatorysequences. A “promotor sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. The promoter is part of theDNA sequence. This sequence region has a start codon at its 3′ terminus.The promoter sequence does include the minimum number of bases whereelements necessary to initiate transcription at levels detectable abovebackground. However, after the RNA polymerase binds the sequence andtranscription is initiated at the start codon (3′ terminus with apromoter), transcription proceeds downstream in the 3′ direction. Withinthe promotor sequence will be found a transcription initiation site(conveniently defined by mapping with nuclease S1) as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

The present invention provides a purified thermostable enzyme thatcatalyzes the removal of arginine, phenylalanine, or methionine aminoacids from the N-terminal end of peptides in peptide or peptidomimeticsynthesis. The purified enzyme is an amidase derived from an organismreferred to herein as “Thermococcus GU5L5” which is a thermophilicarchaeal organism which has a very high temperature optimum. Theorganism is strictly anaerobic and grows between 55 and 90° C.(optimally at 85° C.). GU5L5 was discovered in a shallow marinehydrothermal area in Vulcano, Italy. The organism has coccoid cellsoccurring in singlets or pairs. GU5L5 grows optimally at 85° C. and pH6.0 in a marine medium with peptone as a substrate and nitrogen in gasphase.

The polynucleotide of this invention was originally recovered from agenomic gene library derived from Thermococcus GU5L5 as described below.It contains an open reading frame encoding a protein of 622 amino acidresidues.

In a preferred embodiment, the amidase enzyme of the present inventionhas a molecular weight of about 68.5 kilodaltons as inferred from thenucleotide sequence of the gene.

In accordance with an aspect of the present invention, there areprovided isolated nucleic acid molecules (polynucleotides) which encodefor the mature enzyme having the deduced amino acid sequence of FIG. 1(SEQ ID NO:2).

The deposit is made under the terms of the Budapest Treaty on theInternational Recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and are not an admission that a deposit be required under 35U.S.C. §112. The sequences of the polynucleotides contained in thedeposited materials, as well as the amino acid sequences of thepolypeptides encoded thereby, are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

This invention, in addition to the isolated nucleic acid moleculeencoding an amidase enzyme disclosed in FIG. 1 (SEQ ID NO: 1), alsoprovides substantially similar sequences. Isolated nucleic acidsequences are substantially similar if: (i) they are capable ofhybridizing under stringent conditions, hereinafter described, to SEQ IDNO: 1; or (ii) they encode DNA sequences which are degenerate to SEQ IDNO: 1. Degenerate DNA sequences encode the amino acid sequence of SEQ IDNO:2, but have variations in the nucleotide coding sequences. As usedherein, “substantially similar” refers to the sequences having similaridentity to the sequences of the instant invention. The nucleotidesequences that are substantially similar can be identified byhybridization or by sequence comparison. Enzyme sequences that aresubstantially similar can be identified by one or more of the following:proteolytic digestion, gel electrophoresis and/or microsequencing.

One means for isolating a nucleic acid molecule encoding an amidaseenzyme is to probe a gene library with a natural or artificiallydesigned probe using art recognized procedures (see, for example:Current Protocols in Molecular Biology, Ausubel F. M. et al. (EDS.)Green Publishing Company Assoc. and John Wiley Interscience, New York,1989, 1992). It is appreciated to one skilled in the art that SEQ ID NO:1, or fragments thereof (comprising at least 15 contiguous nucleotides),is a particularly useful probe. Other particular useful probes for thispurpose are hybridizable fragments to the sequences of SEQ ID NO:1(i.e., comprising at least 15 contiguous nucleotides).

With respect to nucleic acid sequences which hybridize to specificnucleic acid sequences disclosed herein, hybridization may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions. As an example of oligonucleotide hybridization, apolymer membrane containing immobilized denatured nucleic acid is firstprehybridized for 30 minutes at 45° C. in a solution consisting of 0.9 MNaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mM Na₂EDTA, 0.5% SDS, 10×Denhardt's,and 0.5 mg/mL polyriboadenylic acid. Approximately 2×10⁷ cpm (specificactivity 4-9×10 cpm/ug) of P end-labeled oligonucleotide probe are thenadded to the solution. After 12-16 hours of incubation, the membrane iswashed for 30 minutes at room temperature in 1×SET (150 mM NaCl, 20 mMTris hydrochloride, pH 7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followedby a 30 minute wash in fresh 1×SET at Tm-10° C. for the oligo-nucleotideprobe. The membrane is then exposed to auto-radiographic film fordetection of hybridization signals.

Stringent conditions means hybridization will occur only if there is atleast 90% identity, preferably at least 95% identity and most preferablyat least 97% identity between the sequences. See J. Sambrook et al.,Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring HarborLaboratory) which is hereby incorporated by reference in its entirety.

“Identity” as the term is used herein, refers to a polynucleotidesequence which comprises a percentage of the same bases as a referencepolynucleotide (SEQ ID NO:1). For example, a polynucleotide which is atleast 90% identical to a reference polynucleotide, has polynucleotidebases which are identical in 90% of the bases which make up thereference polynucleotide and may have different bases in 10% of thebases which comprise that polynucleotide sequence.

The present invention also relates to polynucleotides which differ fromthe reference polynucleotide such that the changes are silent changes,for example the changes do not alter the amino acid sequence encoded bythe polynucleotide. The present invention also relates to nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the enzyme encoded by the referencepolynucleotide (SEQ ID NO:1). In a preferred aspect of the inventionthese enzymes retain the same biological action as the enzyme encoded bythe reference polynucleotide.

It is also appreciated that such probes can be and are preferablylabeled with an analytically detectable reagent to facilitateidentification of the probe. Useful reagents include but are not limitedto radioactivity, fluorescent dyes or enzymes capable of catalyzing theformation of a detectable product. The probes are thus useful to isolatecomplementary copies of DNA from other animal sources or to screen suchsources for related sequences.

The coding sequence for the amidase enzyme of the present invention wasidentified by preparing a Thermococcus GU5L5 genomic DNA library andscreening the library for the clones having amidase activity. Suchmethods for constructing a genomic gene library are well-known in theart. One means, for example, comprises shearing DNA isolated from GU5L5by physical disruption. A small amount of the sheared DNA is checked onan agarose gel to verify that the majority of the DNA is in the desiredsize range (approximately 3-6 kb). The DNA is then blunt ended usingMung Bean Nuclease, incubated at 37° C. and phenol/chloroform extracted.The DNA is then methylated using Eco RI Methylase. Eco R1 linkers arethen ligated to the blunt ends through the use of T4 DNA ligase andincubation at 4° C. The ligation reaction is then terminated and the DNAis cut-back with Eco R1 restriction enzyme. The DNA is then sizefractionated on a sucrose gradient following procedures known in theart, for example, Maniatis, T., et al., Molecular Cloning, Cold SpringHarbor Press, New York, 1982, which is hereby incorporated by referencein its entirety.

A plate assay is then performed to get an approximate concentration ofthe DNA. Ligation reactions are then performed and 1 μl of the ligationreaction is packaged to construct a library. Packaging, for example, mayoccur through the use of purified λgt11 phage arms cut with EcoRI andDNA cut with EcoRI after attaching EcoRI linkers. The DNA and λgt11 armsare ligated with DNA ligase. The ligated DNA is then packaged intoinfectious phage particles. The packaged phages are used to infect E.coli cultures and the infected cells are spread on agar plates to yieldplates carrying thousands of individual phage plaques. The library isthen amplified.

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA or a genomic library to isolate thefull length DNA and to isolate other DNAs which have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype have at least 10, preferably at least 15, and even more preferablyat least 30 bases and may contain, for example, at least 50 or morebases. The probe may also be used to identify a DNA clone correspondingto a full length transcript and a genomic clone or clones that containthe complete gene including regulatory and promotor regions, exons, andintrons.

The isolated nucleic acid sequences and other enzymes may then bemeasured for retention of biological activity characteristic to theenzyme of the present invention, for example, an assay for detectingenzymatic amidase activity. Such enzymes include truncated forms ofamidase, and variants such as deletion and insertion variants.

The polynucleotide of the present invention may be in the form of DNAwhich DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may bedouble-stranded or single-stranded, and if single stranded may be thecoding strand or non-coding (anti-sense) strand. The coding sequencewhich encodes the mature enzyme may be identical to the coding sequenceshown in FIG. 1 (SEQ ID NO:1) and/or that of the deposited clone or maybe a different coding sequence which coding sequence, as a result of theredundancy or degeneracy of the genetic code, encodes the same matureenzyme as the DNA of FIG. 1 (SEQ ID NO:1).

The polynucleotide which encodes for the mature enzyme of FIG. 1 (SEQ IDNO:2) may include, but is not limited to: only the coding sequence forthe mature enzyme; the coding sequence for the mature enzyme andadditional coding sequence such as a leader sequence or a proproteinsequence; the coding sequence for the mature enzyme (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the matureenzyme.

Thus, the term “polynucleotide encoding an enzyme (protein)” encompassesa polynucleotide which includes only coding sequence for the enzyme aswell as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the enzyme having the deduced amino acid sequence of FIG.1 (SEQ ID NO:2). The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature enzyme as shown in FIG. 1 (SEQ ID NO:2) as well as variants ofsuch polynucleotides which variants encode for a fragment, derivative oranalog of the enzyme of FIG. 1 (SEQ ID NO:2). Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO: 1). As known in the art, an allelic variantis an alternate form of a polynucleotide sequence which may have asubstitution, deletion or addition of one or more nucleotides, whichdoes not substantially alter the function of the encoded enzyme.

The present invention also includes polynucleotides, wherein the codingsequence for the mature enzyme may be fused in the same reading frame toa polynucleotide sequence which aids in expression and secretion of anenzyme from a host cell, for example, a leader sequence which functionsto control transporting an enzyme from the cell. The enzyme having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the enzyme. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

Thus, for example, the polynucleotide of the present invention mayencode for a mature enzyme, or for an enzyme having a prosequence or foran enzyme having both a prosequence and a presequence (leader sequence).

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode enzymes which eitherretain substantially the same biological function or activity as themature enzyme encoded by the DNA of FIG. 1 (SEQ ID NO: 1).

Alternatively, the polynucleotide may have at least 15 bases, preferablyat least 30 bases, and more preferably at least 50 bases which hybridizeto a polynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO: 1, for example, for recovery of thepolynucleotide or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% identity and morepreferably at least a 95% identity to a polynucleotide which encodes theenzyme of SEQ ID NO:2 as well as fragments thereof, which fragments haveat least 30 bases and preferably at least 50 bases and to enzymesencoded by such polynucleotides.

The present invention further relates to a enzyme which has the deducedamino acid sequence of FIG. 1 (SEQ ID NO:2), as well as fragments,analogs and derivatives of such enzyme.

The terms “fragment,” “derivative” and “analog” when referring to theenzyme of FIG. 1 (SEQ ID NO:2) means a enzyme which retains essentiallythe same biological function or activity as such enzyme. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature enzyme.

The enzyme of the present invention may be a recombinant enzyme, anatural enzyme or a synthetic enzyme, preferably a recombinant enzyme.

The fragment, derivative or analog of the enzyme of FIG. 1 (SEQ ID NO:2)may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature enzyme is fused withanother compound, such as a compound to increase the half-life of theenzyme (for example, polyethylene glycol), or (iv) one in which theadditional amino acids are fused to the mature enzyme, such as a leaderor secretory sequence or a sequence which is employed for purificationof the mature enzyme or a proprotein sequence. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

The enzymes and polynucleotides of the present invention are preferablyprovided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide or enzymepresent in a living animal is not isolated, but the same polynucleotideor enzyme, separated from some or all of the coexisting materials in thenatural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or enzymes could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment.

The enzymes of the present invention include the enzyme of SEQ ID NO:2(in particular the mature enzyme) as well as enzymes which have at least70% similarity (preferably at least 70% identity) to the enzyme of SEQID NO:2 and more preferably at least 90% similarity (more preferably atleast 90% identity) to the enzyme of SEQ ID NO:2 and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the enzyme of SEQ ID NO:2 and also include portions of suchenzymes with such portion of the enzyme generally containing at least 30amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two enzymes is determined bycomparing the amino acid sequence and its conserved amino acidsubstitutes of one enzyme to the sequence of a second enzyme. Similaritymay be determined by procedures which are well-known in the art, forexample, a BLAST program (Basic Local Alignment Search Tool at theNational Center for Biological Information).

A variant, i.e. a “fragment”, “analog” or “derivative” enzyme, andreference enzyme may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gin, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Most highly preferred are variants which retain the same biologicalfunction and activity as the reference polypeptide from which it varies.

Fragments or portions of the enzymes of the present invention may beemployed for producing the corresponding full-length enzyme by peptidesynthesis; therefore, the fragments may be employed as intermediates forproducing the full-length enzymes. Fragments or portions of thepolynucleotides of the present invention may be used to synthesizefull-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof enzymes of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors containing the polynucleotides of thisinvention. Such vectors may be, for example, a cloning vector or anexpression vector. The vector may be, for example, in the form of aplasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants or amplifying the genes ofthe present invention. The culture conditions, such as temperature, pHand the like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing enzymes by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing an enzyme. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast; plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or tip, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBluescript II (Stratagene); pTRC99a, pKK223-3, pDR540, pRIT2T(Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG,pSVLSV40 (Pharmacia). However, any other plasmid or vector may be usedas long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the enzymes of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the enzymes of the present inventionby higher eukaryotes is increased by inserting an enhancer sequence intothe vector. Enhancers are cis-acting elements of DNA, usually about from10 to 300 bp that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, a cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), at-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated enzyme. Optionally, the heterologoussequence can encode a fusion enzyme including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

Useful expression vector for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEMI (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Coleman,Cell, 23:175 (1981), and host cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The enzyme can be recovered and purified from recombinant cell culturesby methods including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

The enzymes of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the enzymes of the present invention may beglycosylated or may be non-glycosylated. Enzymes of the invention may ormay not also include an initial methionine amino acid residue.

The enzymes, their fragments or other derivatives, or analogs thereof,or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the enzymes corresponding to a sequence ofthe present invention can be obtained by direct injection of the enzymesinto an animal or by administering the enzymes to an animal, preferablya nonhuman. The antibody so obtained will then bind the enzymes itself.In this manner, even a sequence encoding only a fragment of the enzymescan be used to generate antibodies binding the whole native enzymes.Such antibodies can then be used to isolate the enzyme from cellsexpressing that enzyme.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic enzyme products of this invention. Also, transgenic mice maybe used to express humanized antibodies to immunogenic enzyme productsof this invention.

Antibodies generated against the enzyme of the present invention may beused in screening for similar enzymes from other organisms and samples.Such screening techniques are known in the art, for example, one suchscreening assay is described in “Methods for Measuring CellulaseActivities”, Methods in Enzymology, Vol 160, pp. 87-116, which is herebyincorporated by reference in its entirety. Antibodies may also beemployed as a probe to screen gene libraries generated from this orother organisms to identify this or cross reactive activities.

The present invention is further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples retainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotides mayor may not have a 5′ phosphate. Those that do not will not ligate toanother oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide will ligate to afragment that has not been dephosphorylated.

“Ligation” refers to a process of forming phosphodiesternoids betweentwo double stranded nucleic acid fragments (Maniatis et al., Id., p.146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described inthe method of Sambrook, Fritsch and Maniatus, 1989.

EXAMPLE 1 Bacterial Expression and Purification of Amidase

A Thermococcus GU5L5 genomic library was screened for amidase activityas described in Example 2 and a positive clone was identified andisolated. DNA of this clone was used as a template in a 100 μl PCRreaction using the following primer sequences:

5′ primer: CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGACCGGC ATCGAATGGA 3′(SEQ ID NO:3). 3′ primer: 5′ AATAAGGATC CACACTGGCA CAGTGTCAAG ACA 3′(SEQ ID NO:4).

The protein was expressed in E. coli. The gene was amplified using PCRwith the primers indicated above.

Subsequent to amplification, the PCR product was cloned into the EcoRIand BamHI sites of pQET1 and transformed by electroporation into E. coliM15(pREP4). The resulting transformants were grown up in 3 ml cultures,and a portion of this culture was induced. A portion of the uninducedand induced cultures were assayed using Z-L-Phe-AMC (see below).

The primer sequences set out above may also be employed to isolate thetarget gene from the deposited material by hybridization techniquesdescribed above.

EXAMPLE 2 Discovery of an Amidase from Thermococcus GU5L5

Production of the Expression Gene Bank.

Colonies containing pBluescript plasmids with random inserts from theorganism Thermococcus GU5L5 was obtained according to the method of Hayand Short. (Hay, B. and Short, J., Strategies. 1992, 5, 16.) Theresulting colonies were picked with sterile toothpicks and used tosingly inoculate each of the wells of 96-well microtiter plates. Thewells contained 250 μL of LB media with 100 μg/mL ampicillin, 80 μg/mLmethicillin, and 10% v/v glycerol (LB Amp/Meth, glycerol). The cellswere grown overnight at 37° C. without shaking. This constitutedgeneration of the “SourceGeneBank”; each well of the Source GeneBankthus contained a stock culture of E. coli cells, each of which containeda pBluescript plasmid with a unique DNA insert.

Screening for Amidase Activity.

The plates of the Source GeneBank were used to multiply inoculate asingle plate (the “Condensed Plate”) containing in each well 200 μL ofLB Amp/Meth, glycerol. This step was performed using the High DensityReplicating Tool (HDRT) of the Beckman Biomek with a 1% bleach, water,isopropanol, air-dry sterilization cycle in between each inoculation.Each well of the Condensed Plate thus contained 10 to 12 differentpBluescript clones from each of the source library plates. The CondensedPlate was grown for 16 h at 37° C. and then used to inoculate two white96-well Polyfiltronics microtiter daughter plates containing in eachwell 250 μL of LB Amp/Meth (without glycerol). The original condensedplate was put in storage −80° C. The two condensed daughter plates wereincubated at 37° C. for 18 h.

The ‘600 μM substrate stock solution’ was prepared as follows: 25 mg ofN-morphourea-L-phenylalanyl-7-amido-4-trifluoromethylcoumarin(Mu-Phe-AFC, Enzyme Systems Products, Dublin, Calif.) was dissolved inthe appropriate volume of DMSO to yield a 25.2 mM solution. Two hundredfifty microliters of DMSO solution was added to ca. 9 mL of 50 mM, pH7.5 Hepes buffer containing 0.6 mg/mL of dodecyl maltoside. The volumewas taken to 10.5 mL with the above Hepes buffer to yield a cloudysolution.

Fifty μL of the ‘600 μM stock solution’ was added to each of the wellsof a white condensed plate using the Biomek to yield a finalconcentration of substrate of ˜100 μM. The fluorescence values wererecorded (excitation=400 nm, emission=505 nm) on a plate readingfluorometer immediately after addition of the substrate. The plate wasincubated at 70° C. for 60 min. and the fluorescence values wererecorded again. The initial and final fluorescence values weresubtracted to determine if an active clone was present by an increase influorescence over the majority of the other wells.

Isolation of the Active Clone.

In order to isolate the individual clone which carried the activity, theSource GeneBank plates were thawed and the individual wells used tosingly inoculate a new plate containing LB Amp/Meth. As above the platewas incubated at 37° C. to grow the cells, and 50 μL of 600 μM substratestock solution added using the Biomek. Once the active well from thesource plate was identified, the cells from the source plate were usedto inoculate 3 mL cultures of IB/AMP/Meth, which were grown overnight.The plasmid DNA was isolated from the cultures and utilized forsequencing and construction of expression subclones.

EXAMPLE 3 Thermococcus GU5L5 Amidase Characterization

Substrate Specificity.

Using the following substrates (see below for definitions of theabbreviations): CBZ-L-ala-AMC, CBZ-L-arg-AMC, CBZ-L-met-AMC,CBZ-L-phe-AMC, and 7-methyl-umbelliferyl heptanoate at 100 μM for 1 hourat 70° C. in the assays as described in the clone discovery section, therelative activity of the amidase was 3:3:1:<0.1:<0.1 for the compoundsCBZ-L-arg-AMC:CBZ-L-phe-AMC:CBZ-L-met-AMC:CBZ-L-ala-AMC:7-methylumbelliferylheptanoate. The excitation and emission wavelengths for the7-amido-4-methylcoumarins were 380 and 460 nm respectively, and 326 and450 for the methylumbelliferone.

The abbreviations stand for the following compounds:

CBZ-L-ala-AMC=Nα-carbonylbenzyloxy-L-alanine-7-amido-4-methylcoumarin

CBZ-L-arg-AMC=Nα-carbonylbenzyloxy-L-arginine-7-amido-4-methylcoumarin

CBZ-D-arg-AMC=Nα-carbonylbenzyloxy-D-arginine-7-amido-4-methylcoumarin

CBZ-L-met-AMC=Nα-carbonylbenzyloxy-L-methionine-7-amido-4-methylcoumarin

CBZ-L-phe-AMC=Nα-carbonylbenzyloxy-L-phenylalanine-7-amido-4-methylcoumarin

Organic Solvent Sensitivity.

The activity of the amidase in increasing concentrations of dimethylsulfoxide (DMSO) was tested as follows: to each well of a microtiterplate was added 10 μL of 3 mM CBZ-L-phe-AMC in DMSO, 25 μL of celllysate containing the amidase activity, and 250 μL of a variable mixtureof DMSO:pH 7.5, 50 mM Hepes buffer. The reactions were heated for 1 hourat 70° C. and the fluorescence measured. FIG. 2 shows the fluorescenceversus concentration of DMSO. The filled and open boxes representindividual assays.

The activity and enantioselectivity of the amidase in increasingconcentrations of dimethyl formamide (DMF) was tested as follows: toeach well of a microtiter plate was added 30 μL of 1 mM CBZ-L-arg-AMC,CBZ-D-arg-AMC in DMF, 30 μL of cell lysate containing the amidaseactivity, and 240 μL of a variable mixture of DMF:pH 7.5, 50 mM Hepesbuffer. The reactiosn were incubated at RT for 1 hour and thefluorescence measured at 1 minute intervals. FIG. 3 shows the relativeinitial linear rates (increase in fluorescence per min, i.e.,‘activity’) versus concentration of DMF for the more reactiveCBZ-L-arg-AMC.

The initial linear rate (‘activity’) of the L and the D CBZ-arg-AMCsubstrates are shown in Tables 1 and 2 below:

TABLE 1 Activity of the CBZ-L-arg-AMC: Initial Rate, DMF Fl.U./min 0.4% 654 10% 2548 20% 1451 30% 541 40% 345 50% 303 60% 190 75% 81 90% 11

TABLE 2 Activity of the CBZ-D-arg-AMC: Initial Rate, DMF Fl.U./min 0.4% 0.3 10% 10.1 20% 4.6 30% 1.8 40% 0.9 50% 1.2 60% 1.4 75% 0.1 90% 0.1

The above data indicate that the enzyme shows excellent selectivity forthe L, or ‘natural’ enantiomer of the derivatized amino acid substrate.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

4 1869 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR DNA 1 ATG ACC GGC ATC GAATGG AAC CAC GAG ACC TTT TCT AAG TTC GCC TAC 48 Met Thr Tly Ile Glu TrpAsn His Glu Thr Phe Ser Lys Phe Ala Tyr 5 10 15 CTG GGC GAC CCG AGG ATACGG GGA AAC TTA ATC GCG TAC ACC CTG ACG 96 Leu Gly Asp Pro Arg Ile ArgGly Asn Leu Ile Ala Tyr Thr Leu Thr 20 25 30 AAG GCC AAC ATG AAG GAC AACAAG TAC GAG AGC ACG GTT GTT GTT GAA 144 Lys Ala Asn Met Lys Asp Asn LysTyr Glu Ser Thr Val Val Val Glu 35 40 45 GAC CTT GAA ACG GGC TCA AGG CGCTTC ATC GAG AAC GCC TCA ATG CCG 192 Asp Leu Glu Thr Gly Ser Arg Arg PheIle Glu Asn Ala Ser Met Pro 50 55 60 AGG ATT TCG CCA GAC GGC AGA AAG CTCGCC TTC ACC TGC TTT AAC GAG 240 Arg Ile Ser Pro Asp Gly Arg Lys Leu AlaPhe Thr Cys Phe Asn Glu 65 70 75 80 GAG AAG AAG GAG ACC GAG ATA TGG GTGGCC GAT ATC CAG ACC CTG AGC 288 Glu Lys Lys Glu Thr Glu Ile Trp Val AlaAsp Ile Gln Thr Leu Ser 85 90 95 GCC AAG AAA GTC CTC TCA ACT AAA AAC GTCCGC TCG ATG CAG TGG AAC 336 Ala Lys Lys Val Leu Ser Thr Lys Asn Val ArgSer Met Gln Trp Asn 100 105 110 GAC GAT TCA AGG AGA CTC TTA GTT GTC GGCTTC AAG AGG AGG GAC GAT 384 Asp Asp Ser Arg Arg Leu Leu Val Val Gly PheLys Arg Arg Asp Asp 115 120 125 GAG GAC TTC GTC TTT GAC GAC GAC GTC CCGGTC TGG TTC GAC AAT ATG 432 Glu Asp Phe Val Phe Asp Asp Asp Val Pro ValTrp Phe Asp Asn Met 130 135 140 GGA TTC TTT GAT GGA GAG AAG ACG ACG TTCTGG GTT CTT GAC ACT GAG 480 Gly Phe Phe Asp Gly Glu Lys Thr Thr Phe TrpVal Leu Asp Thr Glu 145 150 155 160 GCC GAG GAG ATA ATC GAG CAG TTC GAGAAG CCG AGG TTT TCG AGT GGC 528 Ala Glu Glu Ile Ile Glu Gln Phe Glu LysPro Arg Phe Ser Ser Gly 165 170 175 CTC TGG CAC GGC GAT GCG ATA GTT GTGAAC GTC CCG CAC CGC GAG GGG 576 Leu Trp His Gly Asp Ala Ile Val Val AsnVal Pro His Arg Glu Gly 180 185 190 AGC AAG CCT GCC CTG TTC AAG TTC TACGAC ATA GTC CTA TGG AAG GAC 624 Ser Lys Pro Ala Leu Phe Lys Phe Tyr AspIle Val Leu Trp Lys Asp 195 200 205 GGG GAG GAA GAG AAG CTC TTC GAG AGGGTC TCC TTC GAG GCG GTT GAC 672 Gly Glu Glu Glu Lys Leu Phe Glu Arg ValSer Phe Glu Ala Val Asp 210 215 220 TCC GAC GGA AAG AGA ATA CTC CTG AGGGGC AAG AAA AAA AAG CGG TTC 720 Ser Asp Gly Lys Arg Ile Leu Leu Arg GlyLys Lys Lys Lys Arg Phe 225 230 235 240 ATC AGC GAG CAC GAC TGG CTG TACCTC TGG GAC GGC GAG CTT AAA CCG 768 Ile Ser Glu His Asp Trp Leu Tyr LeuTrp Asp Gly Glu Leu Lys Pro 245 250 255 ATC TAC GAG GGC CCG CTC GAC GTCTGG GAA GCC AAG CTC ACG GAA GGA 816 Ile Tyr Glu Gly Pro Leu Asp Val TrpGlu Ala Lys Leu Thr Glu Gly 260 265 270 AAG GTC TAC TTC CTC ACT CCA GATGCG GGC AGG GTA AAC CTC TGG CTC 864 Lys Val Tyr Phe Leu Thr Pro Asp AlaGly Arg Val Asn Leu Trp Leu 275 280 285 TGG GAC GGG AAG GCC GAG CGT GTTGTT ACC GGC GAC CAC TGG ATT TAC 912 Trp Asp Gly Lys Ala Glu Arg Val ValThr Gly Asp His Trp Ile Tyr 290 295 300 GGG CTT GAC GTC AGC GAT GGC AAAGCA TTG CTC CTC ATC ATG ACC GCC 960 Gly Leu Asp Val Ser Asp Gly Lys AlaLeu Leu Leu Ile Met Thr Ala 305 310 315 320 ACG AGG ATA GGC GAG CTC TACCTC TAC GAC GGC GAG CTG AAA CAG GTC 1008 Thr Arg Ile Gly Glu Leu Tyr LeuTyr Asp Gly Glu Leu Lys Gln Val 325 330 335 ACC GAA TAC AAC GGG CCG ATATTC AGG AAG CTC AAG ACC TTC GAG CCG 1056 Thr Glu Tyr Asn Gly Pro Ile PheArg Lys Leu Lys Thr Phe Glu Pro 340 345 350 AGG CAC TTC CGC TTC AAG AGCAAA GAC CTC GAG ATA GAC GGC TGG TAC 1104 Arg His Phe Arg Phe Lys Ser LysAsp Leu Glu Ile Asp Gly Trp Tyr 355 360 365 CTC AGG CCG GAG GTT AAA GAGGAG AAG GCC CCG GTG ATA GTC TTC GTC 1152 Leu Arg Pro Glu Val Lys Glu GluLys Ala Pro Val Ile Val Phe Val 370 375 380 CAC GGC GGG CCG AAG GGC ATGTAC GGA CAC CGC TTC GTC TAC GAG ATG 1200 His Gly Gly Pro Lys Gly Met TyrGly His Arg Phe Val Tyr Glu Met 385 390 395 400 CAG CTG ATG GCG AGC AAGGGC TAC TAC TGC TGC TTC GTG AAC CCG CGC 1248 Gln Leu Met Ala Ser Lys GlyTyr Tyr Val Val Phe Val Asn Pro Arg 405 410 415 GGC AGC GAC GGC TAT AGCGAA GAC TTC GCG CTC CGC GTC CTG GAG AGG 1296 Gly Ser Asp Gly Tyr Ser GluAsp Phe Ala Leu Arg Val Leu Glu Arg 420 425 430 ACT GGC TTG GAG GAC TTTGAG GAC ATA ATG AAC GGC ATC GAG GAG TTC 1344 Thr Gly Leu Glu Asp Phe GluAsp Ile Met Asn Gly Ile Glu Glu Phe 435 440 445 TTC AAG CTC GAA CCG CAGGCC GAC AGG GAG CGC GTT GGA ATA ACG GGC 1392 Phe Lys Leu Glu Pro Gln AlaAsp Arg Glu Arg Val Gly Ile Thr Gly 450 455 460 ATA AGC TAC GGC GGC TTCATG ACC AAC TGG GCC TTG ACT CAG AGC GAC 1440 Ile Ser Tyr Gly Gly Phe MetThr Asn Trp Ala Leu Thr Gln Ser Asp 465 470 475 480 CTC TTC AAG GCA GGAATA AGC GAG AAC GGC ATA AGC TAC TGG CTC ACC 1488 Leu Phe Lys Ala Gly IleSer Glu Asn Gly Ile Ser Tyr Trp Leu Thr 485 490 495 AGC TAC GCC TTC TCGGAC ATA GGG CTC TGG TAC GAC GTC GAG GTC ATC 1536 Ser Tyr Ala Phe Ser AspIle Gly Leu Trp Tyr Asp Val Glu Val Ile 500 505 510 GGG CCA AAT CCG TTAGAG AAC GAG AAC TTC AGG AAG CTC AGC CCG CTG 1584 Gly Pro Asn Pro Leu GluAsn Glu Asn Phe Arg Lys Leu Ser Pro Leu 515 520 525 TTC TAC GCT CAG AACGTG AAG GCG CCG ATA CTC CTA ATC CAC TCG CTT 1632 Phe Tyr Ala Gln Asn ValLys Ala Pro Ile Leu Leu Ile His Ser Leu 530 535 540 GAG GAC TAC CGC TGTCCG CTC GAC CAG AGC CTT ATG TTC TAC AAC GTG 1680 Glu Asp Tyr Arg Cys ProLeu Asp Gln Ser Leu Met Phe Tyr Asn Val 545 550 555 560 CTC AAG GAC ATGGGC AAG GAA GCC TAC ATA GCG ATA TTC AAG CGC GGC 1728 Leu Lys Asp Met GlyLys Glu Ala Tyr Ile Ala Ile Phe Lys Arg Gly 565 570 575 GCC CAC GGC CACAGC GTC CGC GGA AGC CCG AGG CAC AGG CCG AAG CGC 1776 Ala His Gly His SerVal Arg Gly Ser Pro Arg His Arg Pro Lys Arg 580 585 590 TAC AGG CTC TTCATA GAG TTC TTC GAG CGC AAG CTC AAG AAG TAC GAG 1824 Tyr Arg Leu Phe IleGlu Phe Phe Glu Arg Lys Leu Lys Lys Tyr Glu 595 600 605 GAG GGC TTT GAGGTA GAG AAG ATA CTC AAG GGG AAT GGG AAC TGA 1869 Glu Gly Phe Glu Val GluLys Ile Leu Lys Gly Asn Gly Asn 610 615 620 622 AMINO ACIDS AMINO ACIDLINEAR PROTEIN 2 Met Thr Gly Ile Glu Trp Asn His Glu Thr Phe Ser Lys PheAla Tyr 5 10 15 Leu Gly Asp Pro Arg Ile Arg Gly Asn Leu Ile Ala Tyr ThrLeu Thr 20 25 30 Lys Ala Asn Met Lys Asp Asn Lys Tyr Glu Ser Thr Val ValVal Glu 35 40 45 Asp Leu Glu Thr Gly Ser Arg Arg Phe Ile Glu Asn Ala SerMet Pro 50 55 60 Arg Ile Ser Pro Asp Gly Arg Lys Leu Ala Phe Thr Cys PheAsn Glu 65 70 75 80 Glu Lys Lys Glu Thr Glu Ile Trp Val Ala Asp Ile GlnThr Leu Ser 85 90 95 Ala Lys Lys Val Leu Ser Thr Lys Asn Val Arg Ser MetGln Trp Asn 100 105 110 Asp Asp Ser Arg Arg Leu Leu Val Val Gly Phe LysArg Arg Asp Asp 115 120 125 Glu Asp Phe Val Phe Asp Asp Asp Val Pro ValTrp Phe Asp Asn Met 130 135 140 Gly Phe Phe Asp Gly Glu Lys Thr Thr PheTrp Val Leu Asp Thr Glu 145 150 155 160 Ala Glu Glu Ile Ile Glu Gln PheGlu Lys Pro Arg Phe Ser Ser Gly 165 170 175 Leu Trp His Gly Asp Ala IleVal Val Asn Val Pro His Arg Glu Gly 180 185 190 Ser Lys Pro Ala Leu PheLys Phe Tyr Asp Ile Val Leu Trp Lys Asp 195 200 205 Gly Glu Glu Glu LysLeu Phe Glu Arg Val Ser Phe Glu Ala Val Asp 210 215 220 Ser Asp Gly LysArg Ile Leu Leu Arg Gly Lys Lys Lys Lys Arg Phe 225 230 235 240 Ile SerGlu His Asp Trp Leu Tyr Leu Trp Asp Gly Glu Leu Lys Pro 245 250 255 IleTyr Glu Gly Pro Leu Asp Val Trp Glu Ala Lys Leu Thr Glu Gly 260 265 270Lys Val Tyr Phe Leu Thr Pro Asp Ala Gly Arg Val Asn Leu Trp Leu 275 280285 Trp Asp Gly Lys Ala Glu Arg Val Val Thr Gly Asp His Trp Ile Tyr 290295 300 Gly Leu Asp Val Ser Asp Gly Lys Ala Leu Leu Leu Ile Met Thr Ala305 310 315 320 Thr Arg Ile Gly Glu Leu Tyr Leu Tyr Asp Gly Glu Leu LysGln Val 325 330 335 Thr Glu Tyr Asn Gly Pro Ile Phe Arg Lys Leu Lys ThrPhe Glu Pro 340 345 350 Arg His Phe Arg Phe Lys Ser Lys Asp Leu Glu IleAsp Gly Trp Tyr 355 360 365 Leu Arg Pro Glu Val Lys Glu Glu Lys Ala ProVal Ile Val Phe Val 370 375 380 His Gly Gly Pro Lys Gly Met Tyr Gly HisArg Phe Val Tyr Glu Met 385 390 395 400 Gln Leu Met Ala Ser Lys Gly TyrTyr Val Val Phe Val Asn Pro Arg 405 410 415 Gly Ser Asp Gly Tyr Ser GluAsp Phe Ala Leu Arg Val Leu Glu Arg 420 425 430 Thr Gly Leu Glu Asp PheGlu Asp Ile Met Asn Gly Ile Glu Glu Phe 435 440 445 Phe Lys Leu Glu ProGln Ala Asp Arg Glu Arg Val Gly Ile Thr Gly 450 455 460 Ile Ser Tyr GlyGly Phe Met Thr Asn Trp Ala Leu Thr Gln Ser Asp 465 470 475 480 Leu PheLys Ala Gly Ile Ser Glu Asn Gly Ile Ser Tyr Trp Leu Thr 485 490 495 SerTyr Ala Phe Ser Asp Ile Gly Leu Trp Tyr Asp Val Glu Val Ile 500 505 510Gly Pro Asn Pro Leu Glu Asn Glu Asn Phe Arg Lys Leu Ser Pro Leu 515 520525 Phe Tyr Ala Gln Asn Val Lys Ala Pro Ile Leu Leu Ile His Ser Leu 530535 540 Glu Asp Tyr Arg Cys Pro Leu Asp Gln Ser Leu Met Phe Tyr Asn Val545 550 555 560 Leu Lys Asp Met Gly Lys Glu Ala Tyr Ile Ala Ile Phe LysArg Gly 565 570 575 Ala His Gly His Ser Val Arg Gly Ser Pro Arg His ArgPro Lys Arg 580 585 590 Tyr Arg Leu Phe Ile Glu Phe Phe Glu Arg Lys LeuLys Lys Tyr Glu 595 600 605 Glu Gly Phe Glu Val Glu Lys Ile Leu Lys GlyAsn Gly Asn 610 615 620 50 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAROligonucleotide 3 CGAGAATTC ATTAAAGAGG AGAAATTAAC TATGACCGGC ATCGAATGGA50 33 NUCLEOTIDES NUCLEIC ACID SINGLE LINEAR Oligonucleotide 4AATAAGGATC CACACTGGCA CAGTGTCAAG ACA 33

What is claimed is:
 1. A method for producing a polypeptide comprising:transforming or transfecting a cell with a vector containing: a) apolynucleotide encoding a polypeptide of SEQ ID NO:2; or b) apolynucleotide which encodes a polypeptide that is at least 70%identical to a polypeptide of SEQ ID NO:2 having amidase activity, suchthat the cell expresses the polypeptide encoded by the polynucleotide.2. The method of claim 1, wherein the vector is an expression vector. 3.The method of claim 1, wherein the polynucleotide is set forth in SEQ IDNO:1.
 4. The method of claim 2, wherein the vector is a plasmid.
 5. Themethod of claim 2, wherein the vector is virus-derived.
 6. The method ofclaim 1, wherein the cell is a prokaryote.
 7. The method of claim 1,wherein the cell is a eukaryote.